The goal of this procedure is to determine hepatic glucose production in a polycystic ovary syndrome, or PCOS mouse model. Dysregulated glucose metabolism is an important manifestation of PCOS and is key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance.
In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring the M plus two enrichment of six six deuterated glucose, a stable isotopic glucose tracer, via gas chromatography, mass spectrometry, or GCMS. This procedure of involves creation of a stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. One day before the procedure, prepare the stable isotope glucose tracer in normal saline.
For this experiment, glucose production during fasting in glucose-rich conditions were measured so the glucose isotope was prepared in two different preparations. The tracer was prepared under sterile conditions by dissolving sterile and pyrogen-free six six deuterated glucose and sterile 0.9%sodium chloride solution with or without glucose. The first infusate was prepared with only the tracer.
The second infusate contained both the tracer along with the nonisotopic glucose. Once the solutions were prepared, they were sterile filtered with the 0.22 micron filter and stored at four degrees Celsius. The solutions were prepared in such a way that the final infusing dose was close to one milligram per kilogram and minute and two milligrams per kilogram and minute respectively.
For the glucose appearance rate measurement during basal condition in glucose-rich condition, a primed average constant rate infusion of six six deuterated glucose at 1.08 milligrams per kilogram and minute and 1.9 milligrams per kilogram in minute respectively were used. In order to mimic fed state, we employed an average D-glucose infusion rate of 18.8 milligrams per kilogram and minute during glucose-rich condition. Remove the mice from their home cages, and place them in their fasting cages three hours prior to the start of the experiment.
For this experiment, we used four month-old female mice from the C57BL6J strain. Assemble the caging equipment by grouping the mice into a set number of mice per insulin pump. Place partitions on top of a flat, stable base to make individual stalls for the mice.
Cages are clear and made out of plexiglass. There is a flat, stable base upon which the partitions sit. The door to the cage slides to close and has a notch at the bottom to allow the tail to fit through.
Assemble the syringes containing one milliliter of basal infusate, then connect to infusion pump tubing using the polyethylene tubing. Prepare the infusion pump by setting the rate to 150 microliters an hour, which is the basal rate. Heat a water bath to 48 degrees Celsius.
Prepare the catheter insertion station adjacent to the water bath containing the 30 gauge half-inch needles, silastic tubing, and one-inch, clear transport tape. Select one mouse and place it in a secure holder with access to the tail. Place a piece of tape over the proximal portion of the tail to allow space for the catheter insertion more distally.
Bring the mouse to the water bath, and insert the tail in the water bath for approximately 30 to 45 seconds. This helps to dilate the tail vasculature for catheter placement. The catheter insertion must be done under sterile conditions.
Once the tail's warmed, clean the tail, and place a small copper toothless alligator clamp as a tourniquet at the proximal end of the tail. Visualize the lateral tail vein under a magnifying glass. Then, carefully insert the catheter into the tail vein, and flush the solution gently to ensure patency of the catheter.
Wrap a piece of transport tape around insertion site to secure the catheter, and remove the small tourniquet from the tail. Place the mouse in its individual cage and close the sliding door, ensuring that the tail's protruding through the notch and remains outside of the cage. Place an additional piece of tape over the whole catheter and the tail to secure it to the base plate of the cage.
Disconnect the flush from the tail vein catheter, and place the small clamp on the catheter's silastic tubing to prevent backflow while connecting the infusate line from the pump. Once securely connected, remove the clamp and flush the priming solution which consists of the infusate. Ensure that the solution is clear in the tube and not blood-stained.
Once infusion lines are noted to be functioning properly, remove the cover from the mouse. Place standard bedding around the mouse. Start the infusion with the infusate containing the tracer, and run it for three hours continuously.
For the duration of the infusion, continue to check mice well-being, as well as the infusion lines. Ensure that the infusion tubing is properly secured and that there's no leaks from the line connection points. After the first infusion has completed, stop the infusion and place a clamp on the tubing of the catheter to prevent backflow.
Gently removed the mice from their enclosures without disturbing the catheter in order to collect blood. For this experiment, the mice underwent cheek vein puncture using a four millimeter lancet. Collect approximately 75 microliters of blood into your desired vial.
To deproteinize samples in preparation for mass spectrometry, add approximately 15 microliters of blood to 500 microliters of acetone. Remaining blood can be used to check blood glucose level via glucometer and/or a centrifuge to separate plasma for future hormone assays. Remove the infusate from the syringe pumps by disconnecting the tubing from the syringes, and replace it with the second infusate containing tracer along with glucose.
Repeat previous infusion steps using the glucose-rich isotopic glucose infusion. To reach steady state, run a bolus of the second infusate at 600 microliters an hour for 15 minutes. Note the starting time for each group of cages.
Decrease the infusion rate to 150 microliters an hour to complete the three hours of total infusion time. Repeat blood sampling as previously described. Disconnect infusions, apply pressure at the catheter sites until the bleeding stops, and return the mice to their home cages.
Next, the samples are sent for GCMS. Calculate the isotopic enrichment of the glucose isotope under glucose peak by GCCMS using the pentaacetate derivative. Briefly, this method involves preparation of the pentaacetate derivative of glucose, followed by sample analysis using GCMS in the positive chemical ionization mode.
Selective ion monitoring of the mass-to-charge ratio, 171 to 169, is performed to determine the M plus two enrichment of the glucose isotope. All kinetic measurements are performed assuming steady state conditions. Calculate total plasma glucose appearance rate from the M plus two enrichment of the glucose isotope and plasma using established isotope dilution equations.
Under steady state conditions, it is assumed that the rate of appearance of glucose is equal to the rate of disappearance of glucose. Rate of endogenous glucose production equals total plasma glucose rate minus exogenous glucose. Table one includes the representative results from the study.
All units are expressed as milligram per kilogram and minute. Using previously described isotope dilution equations, total plasma glucose rate was calculated. In the control group, the mean glucose rate of appearance was 19.98 in the fasting state.
In glucose-rich conditions, the glucose rate of appearance was 25.8. Glucose production rate was 19.08 in the fasting state and 8.56 in the glucose-rich state. Abnormal glucose metabolism and homeostasis are common in PCOS.
In disorders of abnormal glucose metabolism, regulation of the suppression of glucose production is compromised leading to hyperglycemia. In this study, we describe a straightforward way to measure the rate of hepatic glucose production in multiple mice at the same time. The critical components of this experiment are the catheter insertion and defining the exact measurements of the glucose isotope and natural glucose in order to adequately perform GCMS.
The advantages of the study is accomplishing the infusion in a minimally invasive fashion due to tail vein catheter insertion, as well as use of an easily attainable, stable glucose tracer. There are some limitations to the study, including the technical demand of the procedure and need for possible additional skills. Overall, we describe a straightforward, accurate way to measure the rate of total hepatic glucose production in a PCOS mouse model.
This technique could serve as the foundation for multiple studies involving glucose metabolism of mouse models.
